Synthetic avian-free egg white substitute and uses thereof

ABSTRACT

In some embodiments, the present disclosure relates to an avian-free egg white composition comprising 45-63% Ovalbumin, 9-15% Ovotransferrin, 0-15% Ovomucoid, 3-5% Ovoglobulin G2, 3-5% Ovoglobulin G3, 2.5-5% Ovomucin, 3-5% Lysozyme, 1-2% Ovoinhibitor, 0.8-1.5% Ovoglycoprotein, 0.6-1.0% Flavoprotein, 0.3-0.8% Ovomacroglobulin, 0.02-0.1% Avidin, and 0.02-0.1% Cystatin. In some embodiments, the composition comprises an edible yeast and one or more of the preceding proteins. In some embodiments, the avian-free egg white further comprises one or more of: flavor enhancers, calcium supplements, added vitamins, and a gelling agent. In some embodiments, the present disclosure pertains to a non-allergenic egg-white composition for human consumption. In some embodiments, the present disclosure pertains to methods of making the avian-free egg-white composition.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 15/562,183, filed on Sep. 27, 2017, which is a U.S. nationalstage application of International Patent Application No.PCT/US2016/024430, filed on Mar. 28, 2016, which claims the benefitunder 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No.62/139,492, filed on Mar. 27, 2015, and U.S. Provisional PatentApplication No. 62/169,128, filed on Jun. 1, 2015, each of which isincorporated herein by reference in its entirety as if fully set forthherein.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to synthetic egg white and egg whiteproducts, such as an egg patty, which comprises all of the proteinspresent in an egg white, but which does not contain unhealthy yolk oryolk components, such as cholesterol. The present invention also relatesto processes for making the synthetic egg white and egg white products.

BACKGROUND OF THE INVENTION

Both the white and yolk of an egg are rich in nutrients (e.g., proteins,vitamins and minerals), with the yolk also containing cholesterol, fatsoluble vitamins, and essential fatty acids. Eggs are a very good sourceof inexpensive, high quality protein. More than half the protein of anegg is found in the egg white along with vitamin B2 and lower amounts offat and cholesterol than the yolk. The whites are rich sources ofselenium, vitamin D, B6, B12 and minerals such as zinc, iron and copper.Egg yolks contain more calories and fat. They are the source ofcholesterol, fat soluble vitamins A, D, E and K and lecithin.

However, it is well known that some characteristics of egg and eggproducts are often cause for concern, and otherwise impede the abilityfor some people to consume food products having egg products therein.For example, many individuals may be unable to, or would prefer not toconsume egg products due to health issues like egg allergies or highcholesterol. Other concerns with consuming egg are associated withculinary preferences (such as, a vegetarian or vegan diet), use ofantibiotics and hormones during poultry production, and diseasesassociated with poultry (such as, for example, bird flu). Additionally,the high cost and/or cost fluctuations in the price of eggs and thecontamination of salmonella carried by eggs have also been a concern offood manufacturers. Therefore, there is a need in the art to reduce oreliminate the content of egg and/or egg-based products in some foodproducts to address these concerns.

SUMMARY OF THE INVENTION

In some embodiments, a synthetic avian-free egg white compositioncomprises: 45-63% Ovalbumin, 9-15% Ovotransferrin, 3-5% Ovoglobulin G2,3-5% Ovoglobulin G3, 2.5-5% Ovomucin, 3-5% Lysozyme, 1-2% Ovoinhibitor,0.8-1.5% Ovoglycoprotein, 0.6-1.0% Flavoprotein, 0.3-0.8%Ovomacroglobulin, 0.02-0.1% Avidin, and 0.02-0.1% Cystatin. In someembodiments, the avian-free egg white composition comprises: 45-63%Ovalbumin, 9-15% Ovotransferrin, 0-15% Ovomucoid, 3-5% Ovoglobulin G2,3-5% Ovoglobulin G3, 2.5-5% Ovomucin, 3-5% Lysozyme, 1-2% Ovoinhibitor,0.8-1.5% Ovoglycoprotein, 0.6-1.0% Flavoprotein, 0.3-0.8%Ovomacroglobulin, 0.02-0.1% Avidin, and 0.02-0.1% Cystatin. In someembodiments, one or more proteins may be omitted to create a specified,desired product.

In some embodiments, the avian-free egg white further comprises flavorenhancers. In some embodiments, yeast or modified yeast may be used toenhance flavor and/or texture of a product. In some embodiments, theavian-free egg white further comprises calcium supplement. In someembodiments, the avian-free egg-white composition comprises addedvitamins In some embodiments, the avian-free egg-white compositionfurther comprises a gelling agent. In some embodiments, theaforementioned composition comprises algal omega-3 fatty acids. In someembodiments, such a composition does not induce an allergic reactionupon ingestion by a subject.

In some embodiments, the present disclosure pertains to an avian-freeegg white substitute composition that includes a plurality ofgenetically modified micro-organisms. In some embodiments, thegenetically modified micro-organisms express a plurality of differentegg white proteins. In some embodiments, at least some of the pluralityof different egg white proteins remain within the genetically modifiedmicro-organisms.

In some embodiments, the genetically modified organisms also express oneor more flavor enhancing proteins. In some embodiments, at least some ofthe one or more flavor enhancing proteins also remain within thegenetically modified micro-organisms.

In some embodiments, the plurality of different egg white proteins areselected from the group consisting of Ovalbumin, Ovotransferrin,Ovoglobulin G2, Ovoglobulin G3, Ovomucin, Lysozyme, Ovoinhibitor,Ovoglycoprotein, Flavoprotein, Ovomacroglobulin, Avidin, Ovomucoid, andCystatin. In some embodiments, the one or more flavor enhancing proteinsare selected from the group consisting of myoglobin, casein, actin,gelatin, collagen, and combinations thereof.

In some embodiments, the present disclosure pertains to a method ofmaking an avian-free egg white substitute comprising expressing each ofthe aforementioned proteins individually in a genetically modifiedorganism. In some embodiments the genetically modified organism is ayeast species. In some embodiments, the yeast may be Sacchromycescerevisiae. In an embodiment, the yeast may be Picchia pastoris. In someembodiments, the method comprises expressing at least one of theaforementioned proteins in bacteria. In some embodiments the bacteria isE. coli.

Since Ovomucoid is suspected to cause the majority of human allergies,the formulation may substitute Ovomucoid with either a gelling agentsuch as pectin or a different serine protease inhibitor which may or maynot be a protein (i.e., may be a metal). Albumin, which comprises 54% ofegg white components, may be produced either via genetically engineeredorganisms or via alternate synthetic pathways.

In an embodiment of the present disclosure, the method furthercomprises, expanding the genetically modified cells expressing each ofthe aforementioned proteins in bioreactors. In some embodiments, themethod comprises harvesting and purifying the expressed proteins.

In some embodiments, the present disclosure pertains to a method ofmaking a protein enhanced nutritional yeast. In some embodiments, genesfor a plurality of the afore-mentioned proteins may be expressed in atleast one yeast organism. In an embodiment, genes for at least one ofthe afore-mentioned protein are expressed in a plurality of yeast. In anembodiment, the method comprises combining the colonies of the yeastexpressing different proteins in proportions to create the desirededible product. In an embodiment, this method requires least amounts ofcellular energy for transcription.

In some embodiments, the method comprises expressing at least one geneencoding a protein selected from the group comprising: Ovalbumin,Ovotransferrin, Ovomucoid, Ovoglobulin G2, Ovoglobulin G3, Ovomucin,Lysozyme, Ovoinhibitor, Ovoglycoprotein, Flavoprotein, Ovomacroglobulin,Avidin, and Cystatin in at least one yeast organism. In an embodiment,the method comprises combining yeast colonies and individuallyexpressing at least one of the aforementioned proteins in proportionssimilar to proportions for each protein found in natural egg-white toform protein-enhanced nutritional yeast. In an embodiment, this methodrequires the least amounts of cellular energy for transcription.

In some embodiments, the present disclosure pertains to methods ofmaking an avian-free egg white substitute by expressing genes in aplurality of genetically modified micro-organisms. In some embodiments,the genes encode a plurality of different egg white proteins. In someembodiments, at least some of the plurality of different egg whiteproteins remain within the genetically modified micro-organisms. In someembodiments, the methods of the present disclosure also include a stepof combining the genetically modified micro-organisms to form theavian-free egg white substitute.

In some embodiments, the genes also express one or more flavor enhancingproteins. In some embodiments, at least some of the one or more flavorenhancing proteins also remain within the genetically modifiedmicro-organisms.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory only,and are not restrictive of the invention, as claimed. In thisapplication, the use of the singular includes the plural, the word “a”or “an” means “at least one”, and the use of “or” means “and/or”, unlessspecifically stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements or components comprising one unit and elements orcomponents that comprise more than one unit unless specifically statedotherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated herein byreference in their entirety for any purpose. In the event that one ormore of the incorporated literature and similar materials defines a termin a manner that contradicts the definition of that term in thisapplication, this application controls.

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich the invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisdisclosure: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th ed., R.Reigers et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991).

The following definitions are set forth to illustrate and define themeaning and scope of the various terms used to describe the inventionherein.

A “nucleic acid or polynucleotide sequence” includes, but is not limitedto, eukaryotic mRNA, cDNA, genomic DNA, and synthetic DNA and RNAsequences, comprising the natural nucleoside bases adenine, guanine,cytosine, thymidine, and uracil. The term also encompasses sequenceshaving one or more modified bases.

A “coding sequence” or “open reading frame” refers to a polynucleotideor nucleic acid sequence which can be transcribed and translated (in thecase of DNA) or translated (in the case of mRNA) into a polypeptide invitro or in vivo when placed under the control of appropriate regulatorysequences. The boundaries of the coding sequence are determined by atranslation start codon at the 5′ (amino) terminus and a translationstop codon at the 3′ (carboxy) terminus. A transcription terminationsequence will usually be located 3′ to the coding sequence. A codingsequence may be flanked on the 5′ and/or 3′ ends by untranslatedregions.

“Exon” refers to that part of a gene which, when transcribed into anuclear transcript, is “expressed” in the cytoplasmic mRNA after removalof the introns or intervening sequences by nuclear splicing.

Nucleic acid “control sequences” or “regulatory sequences” refer totranslational start and stop codons, promoter sequences, ribosomebinding sites, polyadenylation signals, transcription terminationsequences, upstream regulatory domains, enhancers, and the like, asnecessary and sufficient for the transcription and translation of agiven coding sequence in a defined host cell. Examples of controlsequences suitable for eucaryotic cells are promoters, polyadenylationsignals, and enhancers. All of these control sequences need not bepresent in a recombinant vector so long as those necessary andsufficient for the transcription and translation of the desired gene arepresent.

The vector may be any vector which may conveniently be subjected torecombinant DNA procedures, and the choice of vector will often dependon the host cell into which it is to be introduced. Thus, the vector maybe an autonomously replicating vector, i.e., a vector which exists as anextra chromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid. Alternatively, the vector maybe one which, when introduced into a host cell, is integrated into thehost cell genome and replicated together with the chromosome(s) intowhich it has been integrated. The vector is preferably an expressionvector in which the encoding DNA sequence is operably linked toadditional segments required for transcription of the DNA. In general,the expression vector is derived from plasmid or viral DNA, or maycontain elements of both. The vector may further comprise elements, suchas, for example, polyadenylation signals (e.g. from SV40 or theadenovirus 5 Elb region), transcriptional enhancer sequences (e.g. theSV40 enhancer) and translational enhancer sequences (e.g. the onesencoding adenovirus VA RNAs).

“Operably or operatively linked” refers to the configuration of thecoding and control sequences so as to perform the desired function.Thus, control sequences operably linked to a coding sequence are capableof effecting the expression of the coding sequence. A coding sequence isoperably linked to or under the control of transcriptional regulatoryregions in a cell when DNA polymerase will bind the promoter sequenceand transcribe the coding sequence into mRNA that can be translated intothe encoded protein. The control sequences need not be contiguous withthe coding sequence, so long as they function to direct the expressionthereof. Thus, for example, intervening untranslated yet transcribedsequences can be present between a promoter sequence and the codingsequence and the promoter sequence can still be considered “operablylinked” to the coding sequence.

The terms “heterologous” and “exogenous” as they relate to nucleic acidsequences such as coding sequences and control sequences, denotesequences that are not normally associated with a region of arecombinant construct, and/or are not normally associated with aparticular cell. Thus, a “heterologous” region of a nucleic acidconstruct is an identifiable segment of nucleic acid within or attachedto another nucleic acid molecule that is not found in association withthe other molecule in nature. For example, a heterologous region of aconstruct could include a coding sequence flanked by sequences not foundin association with the coding sequence in nature. Another example of aheterologous coding sequence is a construct where the coding sequenceitself is not found in nature (e.g., synthetic sequences having codonsdifferent from the native gene). Similarly, a host cell transformed witha construct which is not normally present in the host cell would beconsidered heterologous for purposes of this invention. “Exogenous gene”or “exogenous coding sequence” refers to a nucleic acid sequence notnaturally present in a particular tissue or cell.

“Exogenous protein” refers to a protein not naturally present in aparticular tissue or cell.

“Endogenous gene” refers to a naturally occurring gene or fragmentthereof normally associated with a particular cell.

The expression products described herein may consist of proteinaceousmaterial having a defined chemical structure. However, the precisestructure depends on a number of factors, particularly chemicalmodifications common to proteins. For example, since all proteinscontain ionizable amino and carboxyl groups, the protein may be obtainedin acidic or basic salt form, or in neutral form. The primary amino acidsequence may be derivatized using sugar molecules (glycosylation) or byother chemical derivatizations involving covalent or ionic attachmentwith, for example, lipids, phosphate, acetyl groups and the like, oftenoccurring through association with saccharides. These modifications mayoccur in vitro, or in vivo, the latter being performed by a host cellthrough posttranslational processing systems. Such modifications mayincrease or decrease the biological activity of the molecule, and suchchemically modified molecules are also intended to come within the scopeof the invention.

Alternative methods of cloning, amplification, expression, andpurification will be apparent to the skilled artisan. Representativemethods are disclosed in Sambrook, Fritsch, and Maniatis, MolecularCloning, a Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory(1989).

Eggs are an important source of nutrition. Since the domestication ofthe chicken, people have been enjoying and nourishing themselves witheggs. Additionally, eggs are an important and versatile ingredient forcooking, as their particular chemical makeup is literally the glue ofmany important baking reactions. Egg and egg protein based products areessential ingredients for the volume, texture, and shelf life of aeratedfood products such as muffins, cakes, cookies, breads, and various otherbaked products. Egg and egg protein based products are also used asingredients in non-aerated food products such as noodles, pastas,dumplings and similar foods to provide the hardness and elasticitycharacteristics of these products, typically to enhance the cookingstability and shelf-life of these products. Other benefits to the use ofegg and egg protein based products include providing smoother dough,where applicable, and providing improved processing tolerances forindustrial scale cooking situations.

However, natural fresh whole poultry eggs are known to be high intriglycerides, high density lipids, and, particularly, cholesterol.Because of these components and their adverse effects on the humancardiovascular system, persons at high risk of cardiovascular diseasehave a continuing need for careful dietary control of the types of foodsconsumed. Additionally, natural fresh whole poultry eggs are known to beprone to bacterial contamination and have been implicated in many casesof food poisoning. Pasteurization of natural eggs has simply not been anavailable solution to the problem since such process treatments ofnatural whole poultry eggs serve to break down the white of the egg,causing it to become thin and runny, and causing the egg to appear oldrather than fresh.

Thus, despite egg being labeled as a good source of nutrition, manypeople may need to avoid consumption of natural eggs or egg-relatedproducts due to religious reasons, for e.g. vegetarians. Individuals mayalso not be able to consume eggs or egg-containing products due tohealth reasons, for e.g., allergies to eggs or egg-containing products.

Egg is one of the most common causes of food allergy. It is estimatedthat most children outgrow egg allergy by the age of five, but somepeople remain allergic for a lifetime. The egg is made up of manydifferent proteins, some of which are allergenic and others which arenot. Most people with an egg allergy are allergic to the egg whiteproteins, and others are allergic to the yolk.

As a consequence of the foregoing problems and the increasing awarenessthereof by the consuming public, there exists a need for a syntheticegg-white composition that is nutritionally at par, if not better, withnatural egg, without components that are known to adversely affectcardiovascular health. Additionally, there is a need for syntheticegg-white composition that is avian-free and appeals to vegetarians,because it is animal free.

Egg white is the common name for the clear liquid (also called thealbumen or the glair/glaire) contained within an egg. In chickens it isformed from the layers of secretions of the anterior section of thehen's oviduct during the passage of the egg. It forms around eitherfertilized or unfertilized egg yolks. The primary natural purpose of eggwhite is to protect the yolk and provide additional nutrition for thegrowth of the embryo (when fertilized). Egg white consists primarily ofabout 90% water into which is dissolved 10% proteins. Chicken egg whiteis about two-thirds of an egg's total weight out of its shell, withnearly 92% of that weight coming from water. The remaining weight of theegg white comes from protein, trace minerals, fatty material, vitamins,and glucose. A raw U.S. large egg white weighs 33 grams with 3.6 gramsof protein, 0.24 grams of carbohydrate and 55 milligrams of sodium. Italso contains about 17 calories and no cholesterol. Egg white is analkaline solution and contains approximately 40 different proteins. Theegg white proteins by percentage, along with their natural functionsare: 54% Ovalbumin (Nutrition); 12% Ovotransferrin (Binds iron), 11%Ovomucoid (Blocks digestive enzymes), 4% Ovoglobulin G2, 4% OvoglobulinG3, 3.5% Ovomucin, 3.4% Lysozyme (Kills bacteria), 1.5% Ovoinhibitor, 1%Ovoglycoprotein, 0.8% Flavoprotein, 0.5% Ovomacroglobulin, 0.05% Avidin(Binds biotin), and 0.05% Cystatin.

Unlike the yolk, which is high in lipids (fats), egg white containsalmost no fat, and carbohydrate content is less than 1%. Egg whitescontain just over 50% of the protein in the egg. Egg white proteins havebecome an important and desirable ingredient to the food industry due totheir functional properties which include gelling, foaming, andemulsification. Egg white is also well recognized as an excellent sourceof nutrition. Additionally, egg white is also a component of certainvaccines, for e.g., influenza vaccine.

In some embodiments, the present disclosure relates to an avian-free eggwhite composition comprising 54% Ovalbumin, 12% Ovotransferrin, 11%Ovomucoid, 4% Ovoglobulin G2, 4% Ovoglobulin G3, 3.5% Ovomucin, 3.4%Lysozyme, 1.5% Ovoinhibitor, 1% Ovoglycoprotein, 0.8% Flavoprotein, 0.5%Ovomacroglobulin, 0.05% Avidin, and 0.05% Cystatin. In some embodiments,the composition further comprises flavoring agents. In some embodiments,the avian-free egg white further comprises calcium supplement. In someembodiments, the avian-free egg-white comprises vitamins In someembodiments, the avian-free egg-white composition further comprises agelling agent. In some embodiments, the compositions disclosed hereinfurther comprise additives. In some embodiments, the additives compriseflavoring agents, metals, protease inhibitors, pectins, vitamins,calcium, magnesium, iron, plant-derived omega-3 fatty acids, andsupplements.

In some embodiments, the present disclosure pertains to an avian-freeegg white substitute composition that includes a plurality ofgenetically modified micro-organisms. In some embodiments, thegenetically modified micro-organisms express a plurality of differentegg white proteins. In some embodiments, at least some of the pluralityof different egg white proteins remain within the genetically modifiedmicro-organisms.

In some embodiments, the genetically modified organisms also express oneor more flavor enhancing proteins. In some embodiments, at least some ofthe one or more flavor enhancing proteins remain within the geneticallymodified micro-organisms.

In some embodiments, the plurality of different egg white proteinsinclude, without limitation, Ovalbumin, Ovotransferrin, Ovoglobulin G2,Ovoglobulin G3, Ovomucin, Lysozyme, Ovoinhibitor, Ovoglycoprotein,Flavoprotein, Ovomacroglobulin, Avidin, Ovomucoid, and Cystatin. In someembodiments, the one or more flavor enhancing proteins include, withoutlimitation, myoglobin, casein, actin, gelatin, collagen, andcombinations thereof.

In some embodiments, the present disclosure pertains to a method ofmaking an avian-free egg white substitute comprising expressing each ofthe aforementioned proteins individually in a genetically modifiedorganism. In some embodiments the genetically modified organism is ayeast species. In some embodiments of the present disclosure,microorganisms are used for making the recombinant proteins thatcomprise the synthetic avian-free egg white composition. In someembodiments, the microorganisms comprise, but are not limited to,Saccharomyces cerevesiae, Picchia pastoris, Escherichia coli,lactobacillus species, and blue-green algae. In some embodiments, thecompositions disclosed herein further comprise additives. In someembodiments, the additives comprise flavoring agents, metals, proteaseinhibitors, pectins, vitamins, calcium, magnesium, iron, plant-derivedomega-3 fatty acids, and supplements.

In some embodiments, the method comprises expressing at least one of theaforementioned proteins in bacteria. Some embodiments the bacteria is E.coli. Since Ovomucoid is suspected to cause the majority of humanallergies, the formulation may substitute Ovomucoid with either agelling agent such as pectin or a different serine protease inhibitorwhich may or may not be a protein (i.e., may be a metal). Albumin, whichcomprises 54% of egg white components, may be produced either viagenetically engineered organisms or via alternate synthetic pathways. Inan embodiment of the present disclosure, the method further comprises,expanding the genetically modified cells in bioreactors. In someembodiment, the method furthermore comprises harvesting and purifyingthe expressed protein. In some embodiments, the method further comprisesmixing the individual proteins in proportions similar to the proportionsfound in natural egg-white composition to form an avian-free egg whitetype compound.

In some embodiments, the present disclosure pertains to a method ofmaking a protein enhanced nutritional yeast. In some embodiments, themethod comprises expressing at least one gene encoding a proteinselected from the group comprising: Ovalbumin, Ovotransferrin,Ovomucoid, Ovoglobulin G2, Ovoglobulin G3, Ovomucin, Lysozyme,Ovoinhibitor, Ovoglycoprotein, Flavoprotein, Ovomacroglobulin, Avidin,and Cystatin in at least one yeast organism. In an embodiment, themethod comprises combining yeast colonies and individually expressing atleast one of the aforementioned proteins to form protein-enhancednutritional yeast. In an embodiment, this method requires the leastamounts of cellular energy for transcription. In some embodiments, theprotein enhanced nutritional yeast comprises edible yeast and one ormore of the following proteins: 45-63% Ovalbumin, 9-15% Ovotransferrin,0-15% Ovomucoid, 3-5% Ovoglobulin G2, 3-5% Ovoglobulin G3, 2.5-5%Ovomucin, 3-5% Lysozyme, 1-2% Ovoinhibitor, 0.8-1.5% Ovoglycoprotein,0.6-1.0% Flavoprotein, 0.3-0.8% Ovomacroglobulin, 0.02-0.1% Avidin, and0.02-0.1% Cystatin.

In some embodiments, the present disclosure pertains to a method ofmaking recombinant edible protein compositions for human and animalconsumption. In some embodiments, the recombinant edible proteincomposition comprises egg-white proteins. In an embodiment, the methodcomprises providing at least one vector comprising a gene encoding atleast one protein selected from the group comprising: Ovalbumin,Ovotransferrin, Ovomucoid, Ovoglobulin G2, Ovoglobulin G3, Ovomucin,Lysozyme, Ovoinhibitor, Ovoglycoprotein, Flavoprotein, Ovomacroglobulin,Avidin, and Cystatin. In some embodiments, the vector is a viral vector.In some embodiments, the method further comprises providing at least onehost cell(s). In some embodiments the host cell(s) is a bacterial cell.In some embodiments, the host cell(s) is a yeast cell. In someembodiments, the method comprises transforming, transfecting, orinfecting the at least one host cell(s) with the aforementioned vectorcomprising a gene encoding at least one protein. In some embodiments,the method comprises a plurality of host cells each transfected,transformed, or infected with a vector encoding at least one proteinsuch that each protein is a different protein. In some embodiments, themethod further comprises providing media to culture the transformed,transfected, or infected host cell(s). In some embodiments, the methodcomprises culturing the transformed or transfected or infected hostcell(s) in the culture media under conditions sufficient to express theat least one protein. In some embodiments, the method comprises the stepof purifying the at least one protein. Purification of the crude proteinmay be achieved using any method known in the art, including, withoutlimitation, affinity chromatography. In some embodiments, the methodcomprises mixing the purified proteins in proportions to generate thedesired edible protein composition.

In some embodiments, the present disclosure pertains to methods ofmaking an avian-free egg white substitute by expressing genes in aplurality of genetically modified micro-organisms. In some embodiments,the genes encode a plurality of different egg white proteins. In someembodiments, at least some of the plurality of different egg whiteproteins remain within the genetically modified micro-organisms. In someembodiments, the methods of the present disclosure also include a stepof combining the genetically modified micro-organisms to form theavian-free egg white substitute.

In some embodiments, the genes also express one or more flavor enhancingproteins. In some embodiments, at least some of the one or more flavorenhancing proteins also remain within the genetically modifiedmicro-organisms.

Gene Isolation and Preparation

Genes were selected from national gene sequence databases and specificgenes were ordered from commercial suppliers. Genes sequences wereconfirmed at university labs. Genes with selected sequences wereinserted into viral plasmids. The viral plasmids were inserted intoyeast and/or E. coli bacteria utilizing standard protocols. Hostcolonies were developed and measured for protein expression levels.Colonies with highest yields were isolated and expanded in bioreactors.Proteins were extracted through centrifugation and processed viaprotocols specific to host organism.

Post Processing

Purified proteins were mixed in proportion by weight to re-createdesired product (Egg white, others). When Saccharomyces cerevisiae wasthe host organism, in some applications, the expressed protein was notseparated from host organism. In these instances, the coloniesexpressing different proteins themselves were mixed and deactivated byheat to create an edible host-expressed protein combination.

Ovotransferrin

Ovotransferrin was the first protein to be expressed due to itschallenging structure. Ovotransferrin is has a very complex quarternarystructure with eight disulfide bonds. Ovotransferrin structure wasidentified from the national gene database:http://www.ncbi.nlm.nih.gov/gene/396241—Chromosome 9; NC_006096.3(4096869.4107617, complement).http://www.ncbi.nlm.nih.gov/nuccore/X02009.1 (mRNA sequence). Oligoswere ordered from Integrated DNA technologies. PCR of the ovotransferrinoligo was performed. After an appropriately-sized fragment was obtained,gel was purified and cloned into TOPO pCR2.1. Several mini-preps ofplasmid DNA were performed & followed by an EcoRI digestion. Agarose gelwas performed and the expected sized band was obtained.

The sequence was inserted into S. cerevisiae using the proceduredescribed herein above. Optimization of S. cerevisiae strains/coloniesfor protein production can be achieved by utilizing standardoptimization procedures.

Application and Advantages

The compositions disclosed herein provide for means to create edibleproteins from single-celled organisms which can be useful in providing avaluable source and supply of nutrition in areas with space and/orarable land constraints. The compositions and methods disclosed hereinare especially advantageous as these use fewer resources thanconventional poultry farming to create similar amounts of edibleproteins. Further, the compositions and methods of making thecompositions disclosed herein generate fewer waste products to formsimilar amounts of proteins as conventional poultry farming. The methodsof making the compositions of the present disclosure use organisms withno known nervous system. Thus compositions of the present disclosurealso circumvent animal cruelty issues. Lastly, the compositions andmethods disclosed herein are particularly advantageous because theseprovide means for making an edible protein with customized amino acidcontent and accessory nutrients.

Additional Embodiments

Reference will now be made to more specific embodiments of the presentdisclosure and experimental results that provide support for suchembodiments. However, Applicants note that the disclosure below is forillustrative purposes and is not intended to limit the scope of theclaimed subject matter in any way.

EXAMPLE 1

pPICZα-A-Ovotransferrin was linearized and transformed into host strainGS115 via electroporation. 5-10 positive clones have been selected andverified by PCR. One clone (Ovotransferrin-2) was chosen for cellculture. During 96 hours of induction by methanol, ovotransferrinappeared to be overexpressed and released into cell media examined bySDS PAGE.

1. Materials

pPicZα-A plasmid (Life Tech); host strain DH5!, GS115 (Life Tech);Protein Marker (Creative BioMart); PVDF transfer membrane (Millipore);SUPER ECL PLUS (APPLYGEN); Acr, Bis, Tris (Sigma-Aldrich); SDS(Amresco); Tyrptone, Yeast Extract (OXOID); PCR tube (Fisher); 0.22″msterile filter and dialysis bag (Millipore); Ni2+IDA (Zoonbio); Agarose(Creative BioMart); DNA/plasmids extraction Kits (AXYGEN); SAC I (NEB);Regular chemical reagents (Sigma Aldrich).

2. Recipe

LB: Trypton 1%, Yeast Extract 0.5%, NaCl 1%, PH 7.0; Low salt LB:Trypton 1%, Yeast Extract 0.5%, NaCl 0.5%, PH 7.0, 1.5% agar (plate),adjust pH to 7.4 using NaOH YPD: Yeast Extract 1%, Trypton 2%, glucose2%, agar 1.5% (plate) YPDS: Yeast Extract 1%, Trypton 2%, glucose 2%,0.1 mol/L Sorbitol, agar 1.5% (plate) BMGY: Yeast extract 1%, Tryptone2%, K Phosphate (pH6.0) 100 mM, YNB 1.34%, Biotin (4×10-5%), Glycerol 1%BMMY: Yeast extract 1%, Peptone 2%, K Phosphate (pH6.0) 100 mM, YNB1.34%, Biotin (4×10-5%), methanol 0.5%

3. Instruments

Primarily used. Allegra 21R (BECKMAN) Biological LP, Mini Protean II,Gel Doc2000 PTC-200(MJ Research) 320-S pH (Mettler Toledo) AR5120 Scale(AHOM S) MultiTemp III water bath, Hofer MV-25 UV (Amersham Pharmacia)IceMaker (SANYO) JY92-2D Sonicator Gene Pulser Xcell (BioRad),NANODROP2000 (Thermo)

EXAMPLE 2 Plasmid Extraction and Linearization

Ovotransferrin plasmid was transformed into DH5α strain and amplified.The extraction of pPICAαA-Ovotransferrin plasmid is ˜10″g, linearized bySac I, DNA electrophoresis in agarose gel is shown in FIG. 1.

EXAMPLE 3 Transformation of pPICZaA-Ovotransferrin into GS115

10 μL linearized pPICZaA-Ovotransferrin were pipetted into 80 μLelectrocompetent cells in a 1.5 mL Eppendorf tube, mixed well thentransferred to a cuvette having a 0.2 cm gap. 310 μL of media was addedand the tubes were prechilled for 5 minutes. Electroporation wasperformed at (1700 V, 8 ms, 2 electroshocks). 1 mL prechilled 1 Msorbitol was added into the cuvette, mixed using pipette and the sampletransferred to 2 μL EP tube and, incubated at 30° C. for 2 hours. 50 μL,100 μL and 200 μL cells were plated onto 100 μg/mL Zeocin YPD media andthe plates were incubated at 30° C. for 48 hours. A single colony waspicked up and grown in 10 mL YPD media (100 μg/mL Zeocin) at 30° C.overnight (180 rpm). Cell culture was streaked overnight onto YPD plateshaving 500 μg/mL, 1 mg/mL, and 2 mg/mL. The single colony (Zeocin) waspicked up and grown in the same media overnight. Six positive cloneswere selected and marked as Ovotransferrin-1, Ovotransferrin-2,Ovotransferrin-3, Ovotransferrin-4, Ovotransferrin-5, Ovotransferrin-6.The clones were amplified by PCR using an AOX primer. The targeted geneshould have a length of ˜2.7 kpb.

EXAMPLE 4 Ovotransferrin Expression

Ovotransferrin-2 single colony was streaked into 9 mL YPD (Zeocin 200μg/mL) and incubated at 30° C. for 24 hours (220 rpm). 350 μL waspipetted into 35 mL BMGY (Zeocin 200 μg/mL) and grown at 30° C. for 22hours (220 rpm). The culture was centrifuged at 10,000 g for 2 minutes.Supernatant was poured off and cell pellets were collected. Yeast pelletwas then re-suspended in 35 mL BMMY and 250 mL was transferred to aflask and grown at 30° C. for 24 hours (220 rpm). Next, the yeast pelletsuspension was inducted by adding methanol for 96 hours. Methanol wasadded every 12 hours to keep methanol at 0.5% (v/v) in the flask.Culture media was sampled every 24 hours, centrifuged at 10,000 g for 2minutes, and supernatant was collected and concentrated using PEG20000.The supernatant was then loaded onto an SDS PAGE.

EXAMPLE 5

1. Overview

Using the pPicZaA-RG008 plasmid as templates, the target gene sequencewas amplified. The target gene was then sub-cloned into ExpressionVector PYE-GAPa for expression. After verification by enzyme digestionand sequencing, 10 μg of PYE-GAPa-RG008 plasmid was digested. Eachenzyme cuts once in the GAP promoter region to linearize the vector. Thepichia GS115 was transformed. Positive clones were identified by PCR,and 10 strains of positive clones were obtained. A couple positiveclones were chosen. Large scale expression can be carried out in shakeflask or by fermentation. Expression condition was optimized. RG008-hisprotein is expressed into the medium. The analysis of the RG008 proteinexpression both from the cells and the medium was carried out thoughSDS-PAGE and WB.

2. Material

pPicZα-A plasmid (Life Tech); host strain DH5α, GS115 (Life Tech);Protein Marker (Creative BioMart); PVDF transfer membrane (Millipore);SUPER ECL PLUS (APPLYGEN); Acr, Bis, Tris (Sigma-Aldrich); SDS(Amresco); Tyrptone, Yeast Extract (OXOID); PCR tube (Fisher); 0.22 μmsterile filter and dialysis bag (Millipore); Ni2+IDA (Creative BioMart);Agarose (Creative BioMart); DNA/plasmids extraction Kits (AXYGEN); SAC I(NEB); and Regular chemical reagents (Sigma Aldrich).

3. Recipe:

LB medium: peptone 1%, Yeast Extract 0.5%, NaCl 1%, agar 1.5%, NaOH pH7.4.

Low-Satl LB medium: peptone 1%, Yeast Extract 0.5%, NaCl 0.5%, agar1.5%, NaOH pH 7.4.

YPD medium: peptone 2%, Yeast Extract 1%, dextrose (D-glucose), agar1.5%.

YPDS medium: peptone 2%, Yeast Extract 1%, dextrose (D-glucose), 0.1mol/L Sorbitol, agar 1.5%.

4. Instruments Primarily Used.

Allegra 21R (BECKMAN); Biological LP, Mini Protean II, Gel Doc2000PTC-200 (MJ Research); 320-S pH (Mettler Toledo) AR5120 Scale (AHOM S);MultiTemp III water bath, Hofer MV-25 UV (Amersham Pharmacia) IceMaker(SANYO); JY92-2D Sonicator; Gene Pulser Xcell (BioRad); and NANODROP2000(Thermo).

5. Description of Experimental Procedure

5.1 PYE-GAPa-RG008 Vector Construction

The target gene sequence was amplified. The target fragment of RG008 wassub-cloned into EcoR I and Not I sites of Expression Vector PYE-GAPa.

5.2 Transformation of PYE-GAPa-RG008 into GS115

10 μL linearized PYE-GAPa-RG008 was pipetted into 80 μL electrocompetentcells in a 1.5 mL EP tube. The cells were mixed well and thentransferred to a cuvette having a 0.2 cm gap. 310 μL media were thenadded and the preparation was prechilled for 5 minutes.

Electroporation (1700V, 8 ms, 2 electroshocks) was then performed. 1 mLof prechilled 1M sorbitol was added into the cuvette and mixed using apipette to transfer the sample to a 2 mL EP tube. The preparation wasthen incubated at 30° C. for 2 hours. 50 μL, 100 μL and 200 μL cellpreparations were then plated onto 100 μg/mL Zeocin YPD media. Theplated preparations were then incubated at 30° C. for 48 hours, afterwhich a single colony was picked up and then grown in 10 mL YPD media(100 μg/mL Zeocin) at 30° C. overnight (180 rpm). A cell culture wasthen streaked overnight onto YPD plates having 500 μg/mL, 1 mg/mL, 2mg/mL Zeocin. The single colony was picked up and grown in the samemedia overnight.

5.3 Positive Clones Identification

Six colonies were selected from the YPD plates and genome DNA wasextracted. When amplified by PCR using an AOX1 primer, the target geneshould have the length of ˜2.6 kb.

5.4 Western Blot Analysis (Supernatant)

Western Blot Identification using Mouse-Anti-His antibody was performed.Expression assessment showed that there was no secrete expression ofPYE-GAPa-RG008 protein (˜79 kDa) in culture supernatant.

5.5 Intracellular Expression Testing

A single colony was streaked into 10 mL YPD and incubated at 30° C.overnight (250 rpm). 0.1 mL was then pipetted into 50 mL YPD and grownat 30° C. for 22 hours (250 rpm). 1 ml of the cell culture wastransferred into a 1.5-ml microcentrifuge tube every hour. These sampleswere used to analyze expression levels and determine an optimal time toharvest. The samples were centrifuged at maximum speed in a table topmicrocentrifuge for 2-3 minutes at room temperature and then poured offsupernatant, collected as cell pellet, and frozen quickly in liquidnitrogen. The cells pellets were then stored at −80° C. until ready toassay.

5.6 Protein Purification by Ni-Affinity Column (Intracellular)

Cell paste harvest from 800 ml Gs115 culture was resuspended in 50 mlBuffer A (20 mM Tris-HCl containing 500 mM NaCl, pH 8.0). Ultrasonicbreaking of cells was performed with protease inhibitors on ice andcentrifuged at 12,000 rpm for 15 minutes. The supernatant was thencollected and loaded onto Ni-NTA column pre-equilibrated by lysisbuffer. The column was washed by a 10 column volume of lysis buffer B,containing 20 mM Tris-HCl, 20 mM imidazole, 150 mM NaCl. The targetproteins were eluted by buffer C (20 mM Tris-HCl buffer, pH 8.0,containing 150 mM NaCl, and 250 mM imidazole). After purification, thetarget protein was transferred into a dialysis bag and loaded onto SDSPAGE.

5.7 Western Blot Analysis (Intracellular)

Using Mouse-Anti-His antibody, one band showed an apparent molecularweight of 70 kDa. The target protein then needed to be verified by theRG008 specify antibody.

EXAMPLE 6

1. Overview

pPICZa-A-Ovotransferrin was linearized and transformed into host strainGS115 via electroporation. 5-10 positive clones were selected andverified by PCR. One clone, Ovotransferrin-2, was chose for cellculture. During 96 hours induction by methanol, ovotransferrin appearsto be overexpressed and released into cell media examined by SDS PAGE.

2. Material

pPicZa-A plasmid (Life Tech); host strain DH5a, GS115 (Life Tech);Protein Marker (Creative BioMart); PVDF transfer membrane (Millipore);SUPER ECL PLUS (APPLYGEN); Acr, Bis, Tris (Sigma-Aldrich); SDS(Amresco); Tyrptone, Yeast Extract (OXOID); PCR tube (Fisher); 0.22 msterile filter and dialysis bag (Millipore); Ni2+IDA (Zoonbio); Agarose(Creative BioMart); DNA plasmids extraction Kits (AXYGEN); SAC I (NEB);and Regular chemical reagents (Sigma Aldrich).

3. Recipe

LB: Trypton 1%, Yeast Extract 0.5%, NaCl 1%, PH 7.0; Low salt LB:Trypton 1%, Yeast Extract 0.5%, NaCl 0.5%, PH 7.0, 1.5% agar (plate),adjust pH to 7.4 using NaOH YPD: Yeast Extract 1%, Trypton 2%, glucose2%, agar 1.5% (plate); YPDS: Yeast Extract 1%, Trypton 2%, glucose 2%,0.1 mol L Sorbitol, agar 1.5% (plate); and BMGY: Yeast extract 1%,Tryptone 2%, K Phosphate (pH6.0) 100 mM, YNB 1.34%, Biotin (4×10-5%),Glycerol 1% BMMY: Yeast extract 1%, Peptone 2%, K Phosphate (pH6.0) 100mM, YNB 1.34%, Biotin (4×10-5%), methanol 0.5%.

4. Instruments

Primarily used: Allegra 21R (BECKMAN); Biological LP, Mini Protean II,Gel Doc2000 PTC-200 (MJ Research); 320-S pH (Mettler Toledo) AR5120Scale (AHOM S)

MultiTemp III water bath, Hofer MV-25 UV (Amersham Pharmacia) IceMaker(SANYO) JY92-2D Sonicator; and Gene Pulser Xcell (BioRad) NANODROP2000(Thermo).

5. Description of Experimental Procedure

5.1. Plasmid Extraction and Linearization

Ovotransferrin plasmid was transformed into DH5a strain and amplified.The extraction of pPICAaA-Ovotransferrin plasmid is −10 g, linearized bySac I, DNA electrophoresis in agarose gel.

5.2. Transformation of pPICZaA-Ovotransferrin into GS115

10 μL of linearized pPICZaA-Ovotransferrin was pipetted into 80 μL ofelectrocompetent cells in a 1.5 mL EP tube and mixed well. The mixturewas then transferred to a cuvette having a 0.2 cm gap, and then 310 μLof media was added to the mixture, which was prechilled for 5 minutes.Electroporation was then performed (1700V, 8 ms, 2 electroshocks). 1 mLof prechilled 1 M sorbitol was added into the cuvette, mixed usingpipette transfer of the sample to a 2 mL EP tube, and incubated at 30°C. for 2 hours. 50 μL, 100 μL, and 200 μL cells were plated onto 100μg/mL Zeocin YPD media. The plates were incubated at 30° C. for 48hours. Next, a single colony was picked up and grown in 10 mL YPD media(100 μg/mL Zeocin) at 30° C. overnight (180 rpm). A cell culture wasstreaked overnight onto YPD plates having 500 μg/mL, 1 mg/mL, 2 mg/mLZeocin. The single colony was picked up and grown in the same mediaovernight. Six positive clones were selected and marked asOvotransferrin-1, Ovotransferrin-2, Ovotransferrin-3, Ovotransferrin-4,Ovotransferrin-5, Ovotransferrin-6. When amplified by PCR using an AOXprimer, the target gene should have the length of −2.7 kpb.

5.3. Ovotransferrin Expression

Ovotransferrin-2 single colony was streaked into 9 mL YPD (Zeocin 200μg/mL), incubated at 30° C. for 24 hours (220 rpm), 350 μL of themixture was pipetted into 35 mL BMGY (Zeocin 200 μg/mL), and grown at30° C. for 22 hours (220 rpm). The culture was centrifuged at 10,000 gfor 2 min, after which supernatant was poured off and cell pellets werecollected. A yeast pellet was re-suspended in a 35 mL BMMY, transferredto a 250 mL flask, grown at 30° C. for 24 hours (220 rpm), andinductioned by adding methanol for 96 hours. Methanol was added every 12hours to keep methanol of 0.5% (v/v) in the flask. Culture media wassampled every 24 hours, centrifuged at 10,000 g for 2 minutes, andsupernatant was collected. The supernatant was concentrated usingPEG20000 and then loaded onto SDS PAGE.

What is claimed is:
 1. An avian-free egg white substitute composition,the composition comprising: a plurality of genetically modifiedmicro-organisms, wherein the genetically modified micro-organismsexpress a plurality of different egg white proteins; and wherein atleast some of the plurality of different egg white proteins remainwithin the genetically modified micro-organisms.
 2. The composition ofclaim 1, wherein the plurality of different egg white proteins areselected from the group consisting of Ovalbumin, Ovotransferrin,Ovoglobulin G2, Ovoglobulin G3, Ovomucin, Lysozyme, Ovoinhibitor,Ovoglycoprotein, Flavoprotein, Ovomacroglobulin, Avidin, Ovomucoid, andCystatin.
 3. The composition of claim 1, wherein the geneticallymodified micro-organisms also express one or more flavor enhancingproteins.
 4. The composition of claim 3, wherein at least some of theone or more flavor enhancing proteins also remain within the geneticallymodified micro-organisms.
 5. The composition of claim 4, wherein the oneor more flavor enhancing proteins are selected from the group consistingof myoglobin, casein, actin, gelatin, collagen, and combinationsthereof.
 6. The composition of claim 1, further comprising a calciumsupplement.
 7. The composition of claim 1, further comprising addedvitamins
 8. The composition of claim 1, further comprising a gellingagent.
 9. The composition of claim 1, further comprising algal omega-3fatty acids.
 10. The composition of claim 1, wherein the geneticallymodified micro-organisms are in the form of edible yeast.
 11. A methodof making an avian-free egg white substitute, the method comprising:expressing genes in a plurality of genetically modified micro-organisms,wherein the genes encode a plurality of different egg white proteins,and wherein at least some of the plurality of different egg whiteproteins remain within the genetically modified micro-organisms; andcombining the genetically modified micro-organisms to form theavian-free egg white substitute.
 12. The method of claim 11, wherein theplurality of different egg white proteins are selected from the groupconsisting of Ovalbumin, Ovotransferrin, Ovoglobulin G2, Ovoglobulin G3,Ovomucin, Lysozyme, Ovoinhibitor, Ovoglycoprotein, Flavoprotein,Ovomacroglobulin, Avidin, Ovomucoid, and Cystatin.
 13. The method ofclaim 11, wherein the genes also encode one or more flavor enhancingproteins.
 14. The method of claim 13, wherein at least some of the oneor more flavor enhancing proteins also remain within the geneticallymodified micro-organisms.
 15. The method of claim 13, wherein the one ormore flavor enhancing proteins are selected from the group consisting ofmyoglobin, casein, actin, gelatin, collagen, and combinations thereof.16. The method of claim 11, wherein the genetically modifiedmicro-organisms comprise multiple yeast colonies.
 17. The method ofclaim 16, wherein the multiple yeast colonies comprise Sacchromycescerevisiae.
 18. The method of claim 11, wherein the genetically modifiedmicro-organisms comprise bacteria.
 19. The method of claim 18, whereinthe bacteria comprise E. coli.